QUANTITATION OF FLUORESCENCE ENERGY-TRANSFER BETWEEN CELL-SURFACE PROTEINS VIA FLUORESCENCE DONOR PHOTOBLEACHING KINETICS

We describe practical aspects of photobleaching fluorescence energy tr ansfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are p resent. Measuring differences in donor photobleaching rates on cells l abeled with donor only (fluorescein isothiocyanate-conjugated proteins ) and with both donor and acceptor (tetramethylrhodamine-conjugated pr oteins) allows calculation of the fluorescence energy transfer efficie ncy. We assess possible methods of data analysis in light of the under lying processes of photobleaching and energy transfer and suggest opti mum strategies for this purpose. Single murine B lymphocytes binding v arious ratios of donor and acceptor conjugates of tetravalent concanav alin A (Con A) and divalent succinyl Con A were examined for interlect in energy transfer by these methods. For Con A, a maximum transfer eff iciency of 0.49 +/- 0.02 was observed. Under similar conditions flow c ytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transf er determinations, we present examples of individual cell data and kin etic analyses, population rate constant distributions, and error estim ates for the various quantities involved.